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The aim of the present study was to compare the acute and cumulative cytotoxicity of intact (n-GE) and warmed genipin (w-GE), while investigating the differences in crosslinking capabilities of these two genipins by rheological and mechanical tests. The n-GE solution was prepared by dissolving genipin powder in a sodium phosphate buffer solution. The w-GE solution was prepared by warming the n-GE solution at 37 °C for 24 h. The mechanical tests for chitosan (CH)/genipin gels showed the crosslinking rate of w-GE was much greater than that of n-GE up until 6 h after preparation, whereas the degree of crosslinking of CH/n-GE gels became higher at 12 h. The ISO 10993-5 standard method, which is established specifically for evaluating cumulative cytotoxicity, determined equivalent IC50 for w-GE (0.173 mM) and n-GE (0.166 mM). On the other hand, custom-made cytotoxicity tests using a WST-8 assay after 1 h of cultivation showed that the acute cytotoxicity of w-GE was significantly higher than that of n-GE at concentrations between 0.1–5 mM. The acute cytotoxicity of w-GE should be taken into consideration in its practical uses, despite the fact that the much faster crosslinking of w-GE is useful as an effective cross linker for in-situ forming gels.

Takefumi Narita,1 Shunji Yunoki,1 Yoshimi Ohyabu,1 Naohisa Yahagi,2 Toshio Uraoka3 1Biotechnology Group, Tokyo Metropolitan Industrial Technology Research Institute, Koto-ku, 2Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University School of Medicine, Shinjuku-ku, 3Department of Gastroenterology, National Hospital Organization Tokyo Medical Center, Meguro-ku, Tokyo, Japan Abstract: We investigated the potential of collagen–genipin sols as biomaterials for treating artificial ulcers following endoscopic submucosal dissection. Collagen sol viscosity increased with condensation, allowing retention on tilted ulcers before gelation and resulting in collagen gel deposition on whole ulcers. The 1.44% collagen sols containing genipin as a crosslinker retained sol fluidity at 23°C for >20min, facilitating endoscopic use. Collagen sols formed gel depositions on artificial ulcers in response to body temperature, and high temperature responsiveness of gelation because of increased neutral phosphate buffer concentration allowed for thick gel deposition on tilted ulcers. Finally, histological observations showed infiltration of gels into submucosal layers. Taken together, the present data show that genipin-induced crosslinking significantly improves the mechanical properties of collagen gels even at low genipin concentrations of 0.2–1mM, warranting the use of in situ gelling collagen–genipin sols for endoscopic treatments of gastrointestinal ulcers. Keywords: collagen, genipin, in-situ gel, fibril formation, ulcer dressing&nbsp

Progress in stem cell research has prioritized the refinement of cell-labeling techniques for in vitro and in vivo basic and therapeutic studies. Although quantum dots, because of their optical properties, are emerging as favorable nanoparticles for bioimaging, substantial refinements or modifications that would improve their biocompatibility are still required. We report here that internalizing quantum dots (i-QDs) generated by their conjugation with an internalizing antibody against a heat shock protein-70 family stress chaperone, mortalin, offered an efficient, genetically noninvasive, nontoxic, and functionally inert way to label mesenchymal stem cells (MSCs). The i-QD-labeled MSCs underwent normal adipocyte, osteocyte, and chondrocyte differentiation in vitro and in vivo, suggesting the potential application of i-QDs in in vivo diagnostics, regenerative and therapeutic medicine.

Our objective was to examine the technique of regenerating cartilage tissue from bone marrow-derived cells by three-dimensional (3D) culture using the rotating wall vessel (RWV) bioreactor. Three-dimensional and cylindrical aggregates of allogeneic cartilage with dimensions of 10 x 5 mm (height x diameter) formed by the RWV bioreactor were transplanted into osteochondral defects of Japanese white rabbits (Group T, n = 15). For the control, some osteochondral defects were left empty (Group C, n = 18). At 4, 8, and 12 weeks postimplantation, the reparative tissues were evaluated macroscopically, histologically, and biochemically. In Group T at as early as 4 weeks, histological observation, especially via safranin-O staining, suggested that the reparative tissues resembled hyaline cartilage. And we observed no fibrous tissues between reparative tissue and adjacent normal tissues. In the deeper portion of the bony compartment, the osseous tissues were well remodeled. At 4 and 8 weeks postimplantation, the mean histological score of Group T was significantly better than that of Group C (p < 0.05). The glycosaminoglycans (GAG)/DNA ratio in both groups increased gradually from 4 to 8 weeks and then decreased from 8 to 12 weeks. We herein report the first successful regeneration of cartilage in osteochondral defects in vivo using allogeneic cartilaginous aggregates derived from bone marrow-derived cells by 3D culture using the RWV bioreactor.

We investigated the temperature-responsive gelation of collagen/genipin solutions using pepsin-solubilized collagen (PSC) and acid-solubilized collagen (ASC) as substrates. Gelation occurred in the PSC/genipin solutions at genipin concentrations 0–2 mM under moderate change in temperature from 25 to 37°C. The PSC/genipin solutions exhibited fluidity at room temperature for at least 30 min, whereas the ASC/genipin solutions rapidly reached gel points. In specific cases PSC would be preferred over ASC as an injectable gel system. The temperature-responsive gelation of PSC/genipin solutions was due to temperature responses to genipin crosslinking and collagen fibril formation. The elastic modulus of the 0.5% PSC/genipin gel system could be adjusted in a range of 2.5 to 50 kPa by the PSC and genipin concentrations, suggesting that a PSC/genipin solution is a potential injectable gel system for drug and cell carriers, with mechanical properties matching those of living tissues.

Shunji Yunoki,1 Masushi Kohta,2 Yoshimi Ohyabu,1 Tetsuji Iwasaki2 1Biotechnology Group, Tokyo Metropolitan Industrial Technology Research Institute, 2Medical Engineering Laboratory, ALCARECo., Ltd, Tokyo, Japan Purpose: This study evaluated the in vitro antibacterial activity and cytotoxicity of various commercially available silver-containing dressings (Ag dressing). Methods: Biohesive Ag (hydrocolloid, silver sulfadiazine), Aquacel® Ag (nonwoven fabric, ionic silver [Ag+]), Algisite™ Ag (nonwoven fabric, Ag+), Mepilex® Ag (foam, silver sulfate), and PolyMem® Ag (foam, nanocrystalline silver) were tested for characteristics of Ag+ release, antibacterial activity, and cytotoxicity. The release of Ag+ was investigated in cell culture medium at immersion periods of 6, 24, and 48 hours. The antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa were accessed by a disc diffusion test. The cytotoxicity was evaluated using V79 cells, by an extraction method. Results: The cytotoxicity was not a monotonic function of the antibacterial activity among the Ag dressings and could not be simply explained by Ag+-release properties. Biohesive Ag was regarded as a slow-release Ag dressing, showing the lowest cytotoxicity, while the antibacterial activity was classified as “strong” or “significant” against the two species of bacteria. Aquacel Ag and Algisite Ag showed higher antibacterial activity and cytotoxic effects, which were supported by the higher Ag+ release. Mepilex Ag showed the highest release of Ag+, and the cytotoxicity was the highest among the Ag dressings. However, the antibacterial activity was classified as “significant” or “no activity” for P. aeruginosa and S. aureus, respectively. PolyMem Ag showed the lowest Ag+ release, and the antibacterial activity classified as “significant" or "no activity” for S. aureus and P. aeruginosa, respectively, whereas the cytotoxicity was similar to those of Aquacel Ag and Algisite Ag. Conclusion: The efficacy and adverse effects of the Ag dressings revealed differences that should be considered by clinicians during wound management. Keywords: ionic silver, silver sulfadiazine, nanocrystalline silver, wound management, wound infection, slow release